High conductance Ca2+ and voltage activated K+ channels (Slo1 or BK channels) are widely distributed and play numerous physiological roles. BK channels function as ? subunits alone, as in skeletal muscle, or in association with auxiliary ? subunits (?1- ?4) where they confer diverse functional properties in different tissues. Defectve or missing BK channels or their ? subunits have been associated with many disease processes including hypertension, asthma, autism, mental retardation, obesity, and epilepsy. Understanding the normal mechanism of activation of BK channels is crucial to understanding how function is altered in disease, and to provide molecular information that would be useful in developing possible therapies. The four ? subunits of Slo1 assemble to form a channel with an intra-membrane Core and a cytoplasmic Tail. The Core consists of four voltage sensing domains (VSD) and a pore gate domain (PGD). The cytoplasmic Tails form a large intracellular gating ring. Ca2+ binding to the gating ring activates the PGD through a poorly understood coupling mechanism from gating ring to Core. Also poorly understood are the sites and mechanisms of action of the various ? subunits on BK channels. Two recent advances will allow us to apply new approaches to resolve the mechanisms of coupling and ? subunit action. The first advance is obtaining the protein crystal structures of the gating ring in the closed and open conformations, which suggests that Ca2+ binding to the gating ring induces a push to the Core under the VSDs resulting from elevation of the four alpha-B helices of the gating ring, and a simultaneous pull on the S6 segments in the PGD of the Core arising from movement of lever arms in the gating ring. The second advance was our isolation and functional expression of the isolated Core itself, which provides a tool to assign observed functions to Core, gating ring, or both. Based on these advances and functional data we hypothesize that a novel Push-Pull mechanism couples Ca2+-dependent activation from the gating ring to the Core. In Aim 1 we critically test the Push-Pull hypothesis for Ca2+-dependent coupling using mutations with expected outcomes based on the Push-Pull hypothesis. In Aim 2 we use these advances to localize the sites of action of ?1- ?4 subunits on modifying gating of BK channels to the Core, gating ring, or both. In Aim 3 we seek to obtain the protein crystal structures of the mutated gating rings that alter Ca2+-dependent coupling and also the structures of the sites of contact between peptides of ? subunits and gating ring to provide structural insight into mechanism. The completion of these aims should provide new insight into the mechanism of Ca2+-dependent coupling between gating ring and Core, and also into the mechanisms for ? subunit modulation of BK channels. The Push-Pull model, if found to be consistent with the critical tests to be applied, will necessitate a paradigm shift in the proposed mechanism of Ca2+-dependent coupling, from a single active coupling structure to dual simultaneously active Push-Pull coupling structures.